Fluorescent Gold Nanoclusters for Biosensor and Bioimaging Application

: With the rapid development of materials technology, ﬂuorescent gold nanoclusters (AuNCs) are emerging as novel functional materials for diagnostic applications including the detection of biomarkers and bioimaging due to the advantages of their ultra-small size, tunable emissions, size-dependent ﬂuorescence and excellent biocompatibility. In this review, we introduced the synthetic methods, and physical and chemical properties of AuNCs. Subsequently, we described the AuNCs-based design strategies for the detection of biomarkers including small molecules, DNA and proteins. The applications of AuNCs for tumor imaging in vitro and in vivo were also presented. Finally, we discussed the challenges and potential solutions of AuNCs-based nanosensors.


Introduction
A growing number of diseases are seriously endangering human health, and early diagnosis is exceedingly crucial to improve the condition of patients [1][2][3]. Biomarkers, which are usually found in body fluids including urine or blood, have always been considered as indicators of specific diseases. Rapid, sensitive and cost-effective biosensors for the detection of biomarkers are in high demand, to meet the needs of clinical testing and scientific research [4][5][6]. The development of special nanomaterials such as fluorescent tags in the last decade is an attractive field due to their potential applications in life sciences [7][8][9][10]. Fluorescent nanoclusters (NCs), such as Au, Ag, Pt, Cu, etc., as fluorophores have drawn increasing attention due to their distinctive chemical and physical properties [11][12][13]. Particularly, fluorescent gold nanoclusters (AuNCs), with their size-dependent chemical, electrical and optical properties, have been widely explored for their fundamental scientific research and diverse biological applications [14][15][16]. AuNCs are ultra-small particles consisting of several to a few hundred gold atoms. Due to their size approaching the Fermi-wavelength of electrons, between Au atoms and nanoparticles, AuNCs exhibit molecule-like characteristics, such as discrete energy levels, size-dependent fluorescence and strong photoluminescence. Along with the perfect properties of a large Stokes shift, long life-time, good photostability and biocompatibility, AuNCs are suitable candidates for fluorescent probes, for advancing biological applications such as biosensors and molecular imaging [17][18][19]. Although some reviews have demonstrated the utility of fluorescent AuNCs in sensing and/or imaging, ideas on AuNCs specifically targeting disease-related biomarkers are not well organized.

AuNCs-Based Fluorescent Sensors for Detection of DNA and MicroRNA
Hosseini et al. showed a fluorescence turn-on biosensor for the detection of microRNA [29]. AuNCs were synthesized by templated DNA, containing the complementary sequence of target Crystals 2020, 10, 357 4 of 12 microRNA and two cytosine rich sequence fragments at the 3 and 5 ends. The addition of microRNA-21 led to an increased fluorescence intensity. This sensor had a detection limit of 0.7 pM. Wang et al. reported a fluorescent biosensing platform for DNA detection through the combination of DNA-templated AuNCs (DNA-AuNCs) and MnO 2 sheets [30]. This strategy was based on the specific binding of DNA-AuNCs probe towards targets and the different adsorption between DNA structures and MnO 2 . The detection limit of this assay was 0.2 nM DNA. Similarly, when using aptamer as affinity ligands, an AuNCs-based fluorescent sensor could be used for thrombin detection.

AuNCs-Based Fluorescent Sensors for Detection of Small Molecules with Biological Activity
AuNCs-based biosensors for the detection of small biological molecules had been achieved using various design strategies. Mao reported a ratiometric fluorescent probe for sensing glucose, one of the most significant biomolecules in the biological systems [31]. Ovalbumin protected AuNCs (Ova-AuNCs), with good emission and perfect biocompatibility was used as fluorescent tag. Poly(Nacryloxysuccinimide)-aminophenyl boronic acid-Alizarin Red S (PNAS-APBA-ARS) was used as the response signal probe and specific recognition element for the detection of the target. The detection of this biosensor was 0.1 mM glucose. Sha et al. reported a fluorescence biosensor for the sensitive detection of 5-hydroxytryptamine (5-HT), using transferrin-encapsulated AuNCs [32]. As shown in Figure 1, the presence of 5-HT induced an aggregation-enhanced emission of transferrin-AuNCs due to the high affinity between sialic acid residues of transferrin and 5-HT. The changing of the fluorescence signal was used for the quantitative detection of 5-HT, in the range from 0.2 to 50 µM with a detection limit of 0.049 µM. Zhang et al. presented histidine-templated AuNCs for the fluorescence sensing of Glutathione (GSH). The addition of GSH could induce an enhancement of the fluorescence intensity of AuNCs, enabling the quantitative detection of the target with a detection limit of 25 µM [52]. Vitamins, associated with many metabolic processes of sugars and proteins, play a vital role in human health. Liu et al. displayed a fluorescence sensor for the quantitative detection of Vitamin B 1 , using glutenin-templated AuNCs [53]. Target vitamin could be adsorbed on the surface of glutenin-AuNCs with numerous hydroxyl and carboxyl groups, and fluorescence-quenching signals were measured for sensing targets with a detection limit of 115 nM. BSA-stabilized AuNCs were used for sensing cysteine (Cys) and homocysteine (Hcy) [33]. In this approach, the fluorescence of BSA-AuNCs was quenched by potassium triiodide. In the presence of target Cys and Hcy, the fluorescence recovered through the removement of iodine. The turn-on response enabled the sensitive detection of target molecules. Cui et al. used the BSA-AuNCs-based nanoplatform for the detection of Cys [34]. After the addition of Cys, the forming of Cys-AuNCs complexes enhanced the fluorescence signal, allowing for the quantitative detection of Cys, with a detection limit of 1.2 nM.
Our group reported pH-sensitive AuNCs protected by GSH, for the sensitive detection of Cys by chemical etching [35]. AuNCs, presented in an acidic solution with excess Au(I)-thiolate complexes, showed a pH-sensitive permeability for target Cys, which affected the access of Cys to the embedded AuNCs. Cys enhanced the emission of AuNCs; however, Cys at higher concentrations etched the AuNCs, which induced fluorescence quenching, showing an increased pH signal. This sensor exhibited an ultra-wide linear concentration range from 10 pM to 2 mM and an ultra-low detection limit of 6.3 pM. In addition, we described a novel BSA-AuNCs based fluorescent sensor for the label-free, separation-free and selective detection of Cys [36]. The presence of target Cys could cause etching-induced fluorescence quenching of BSA-AuNCs, allowing the sensitive detection of Cys in the range from 0.5 to10 µM. The detection limit for Cys was 0.15 µM. Our group also presented a chemical etching of an AuNCs-based ratiometric fluorescent sensor for the sensitive detection of tris(2-carboxyethyl)phosphine (TCEP) [37]. The fluorescence spectrum indicated that TCEP could quench the red fluorescent emission of BSA-AuNCs and restore the blue fluorescent emission. The ratio of the blue and red fluorescence intensity of BSA-AuNCs allowed for the quantitative detection of TCEP, with a detection limit of 130 nM. Our group reported pH-sensitive AuNCs protected by GSH, for the sensitive detection of Cys by chemical etching [35]. AuNCs, presented in an acidic solution with excess Au(I)-thiolate complexes, showed a pH-sensitive permeability for target Cys, which affected the access of Cys to the embedded AuNCs. Cys enhanced the emission of AuNCs; however, Cys at higher concentrations etched the AuNCs, which induced fluorescence quenching, showing an increased pH signal. This sensor exhibited an ultra-wide linear concentration range from 10 pM to 2 mM and an ultra-low detection limit of 6.3 pM. In addition, we described a novel BSA-AuNCs based fluorescent sensor for the label-free, separation-free and selective detection of Cys [36]. The presence of target Cys could cause etching-induced fluorescence quenching of BSA-AuNCs, allowing the sensitive detection of Cys in the range from 0.5 to10 μM. The detection limit for Cys was 0.15 μM. Our group also presented a chemical etching of an AuNCs-based ratiometric fluorescent sensor for the sensitive detection of tris(2-carboxyethyl)phosphine (TCEP) [37]. The fluorescence spectrum indicated that TCEP could quench the red fluorescent emission of BSA-AuNCs and restore the blue fluorescent emission. The ratio of the blue and red fluorescence intensity of BSA-AuNCs allowed for the quantitative detection of TCEP, with a detection limit of 130 nM.
Lin et al. demonstrated an AuNCs-based turn-on sensor for GSH detection [38]. The fluorescence of BSA-AuNCs was quenched by MnO2, and in the presence of GSH, MnO2 was digested and the recovered fluorescence was measured to determine the GSH concentration. Yan et al. presented a ratiometric biosensor for the detection of hydrogen sulfide (H2S) [39]. BSA-templated AuNCs were used as the internal reference fluorescent tag and HSip-1 acted as the recognition element and signal indicator. The addition of target H2S enhanced the fluorescence of HSip-1 without affecting the change in BSA-AuNCs fluorescence. Subsequently, the change of ratiometric signal enabled the sensing of the target molecular, with a detection limit of 0.73 μM.

AuNCs-Based Biosensor for Detection of Proteins
Nguyen et al. presented a turn-on fluorescent AuNCs-graphene oxide (GO) nanosensor for the detection of matrix metalloproteinase-9 (MMP-9) [41]. As illustrated in Figure 2, the AuNCs were synthesized by using peptides and mercaptoundecanoic acid (MUA) as co-templating ligands. The Lin et al. demonstrated an AuNCs-based turn-on sensor for GSH detection [38]. The fluorescence of BSA-AuNCs was quenched by MnO 2 , and in the presence of GSH, MnO 2 was digested and the recovered fluorescence was measured to determine the GSH concentration. Yan et al. presented a ratiometric biosensor for the detection of hydrogen sulfide (H 2 S) [39]. BSA-templated AuNCs were used as the internal reference fluorescent tag and HSip-1 acted as the recognition element and signal indicator. The addition of target H 2 S enhanced the fluorescence of HSip-1 without affecting the change in BSA-AuNCs fluorescence. Subsequently, the change of ratiometric signal enabled the sensing of the target molecular, with a detection limit of 0.73 µM.

AuNCs-Based Biosensor for Detection of Proteins
Nguyen et al. presented a turn-on fluorescent AuNCs-graphene oxide (GO) nanosensor for the detection of matrix metalloproteinase-9 (MMP-9) [41]. As illustrated in Figure 2, the AuNCs were synthesized by using peptides and mercaptoundecanoic acid (MUA) as co-templating ligands. The peptide contained an MMP-9 cleavage site. The AuNCs were then loaded to GO, with excellent quenching properties. In the presence of the target enzyme, peptide-AuNCs-GO nanocomplex yielded an increasing fluorescent signal related to the enzyme MMP-9 concentration. The detection limit of this biosensor was 2.5 ng/mL. Liu et al. reported an AuNCs-based fluorescent sensor for the label-free and sensitive detection of protein kinase (PKA) [42]. This approach was based on the Eu 3+ -modulated fluorescence of peptide-templated AuNCs and the PKA-catalyzed hydrolysis. This turn-on method showed a detection limit of 0.02 U/mL PKA. Song developed a peptide-AuNCs-based fluorescence sensor for monitoring PKA detection [43]. The fluorescence quenching of AuNCs occurred due to the cleavage of peptide by carboxypeptidase, without peptide phosphorylation. The adding of PKA induced the phosphorylation of peptide, therefore, the phosphate group could block the digestion of carboxypeptidase, which prevented the fluorescence quenching of the peptide-AuNCs. The measurement of fluorescence intensity change allowed for the detection of PKA. modulated fluorescence of peptide-templated AuNCs and the PKA-catalyzed hydrolysis. This turnon method showed a detection limit of 0.02 U/mL PKA. Song developed a peptide-AuNCs-based fluorescence sensor for monitoring PKA detection [43]. The fluorescence quenching of AuNCs occurred due to the cleavage of peptide by carboxypeptidase, without peptide phosphorylation. The adding of PKA induced the phosphorylation of peptide, therefore, the phosphate group could block the digestion of carboxypeptidase, which prevented the fluorescence quenching of the peptide-AuNCs. The measurement of fluorescence intensity change allowed for the detection of PKA. GSH-stabilized AuNCs were used for the fluorescence detection of trypsin [44]. The fluorescence of GSH-AuNCs could be quenched by the addition of cytochrome c, through the electron transfer mechanism. The presence of trypsin induced the hydrolysis of cytochrome c. Thereby, a significant fluorescence recovery was observed, and the enhanced fluorescence signal enabled the detection of trypsin, with a detection limit of 0.08 μg/mL and a dynamic detection range from 0.2 to 100 μg/mL. As shown in Figure 3, these GSH-AuNCs could be integrated into portable test strips, which show promising potential for point-of-care detection. GSH-stabilized AuNCs were used for the fluorescence detection of trypsin [44]. The fluorescence of GSH-AuNCs could be quenched by the addition of cytochrome c, through the electron transfer mechanism. The presence of trypsin induced the hydrolysis of cytochrome c. Thereby, a significant fluorescence recovery was observed, and the enhanced fluorescence signal enabled the detection of trypsin, with a detection limit of 0.08 µg/mL and a dynamic detection range from 0.2 to 100 µg/mL. As shown in Figure 3, these GSH-AuNCs could be integrated into portable test strips, which show promising potential for point-of-care detection. modulated fluorescence of peptide-templated AuNCs and the PKA-catalyzed hydrolysis. This turnon method showed a detection limit of 0.02 U/mL PKA. Song developed a peptide-AuNCs-based fluorescence sensor for monitoring PKA detection [43]. The fluorescence quenching of AuNCs occurred due to the cleavage of peptide by carboxypeptidase, without peptide phosphorylation. The adding of PKA induced the phosphorylation of peptide, therefore, the phosphate group could block the digestion of carboxypeptidase, which prevented the fluorescence quenching of the peptide-AuNCs. The measurement of fluorescence intensity change allowed for the detection of PKA. GSH-stabilized AuNCs were used for the fluorescence detection of trypsin [44]. The fluorescence of GSH-AuNCs could be quenched by the addition of cytochrome c, through the electron transfer mechanism. The presence of trypsin induced the hydrolysis of cytochrome c. Thereby, a significant fluorescence recovery was observed, and the enhanced fluorescence signal enabled the detection of trypsin, with a detection limit of 0.08 μg/mL and a dynamic detection range from 0.2 to 100 μg/mL. As shown in Figure 3, these GSH-AuNCs could be integrated into portable test strips, which show promising potential for point-of-care detection. The fluorescence enhancement was usually triggered by bio-thiols, due to the AuNCs etch by thiols. Jiang et al. reported a cytidine stabilized AuNCs (Cyt-AuNCs) for sensing GSH, the enhancement of fluorescence intensity enabled the quantitative detection of targets, with a detection limit of 2.0 nM. This platform also allowed for the determination of glutathione reductase (GR) activity, through controlling the production of GSH [45]. Ni et al. showed an AuNCs-based dual-channel assay for turn-on fluorescent and colorimetric sensing of alkaline phosphatase (ALP) [46]. Colorless 3,3 ,5,5 -tetramethylbenzidine (TMB) was oxidized into blue product (oxTMB) by BSA-AuNCs with peroxidase-like activity. The production of oxTMB induced the quenching effect of the AuNCs. The addition of ALP, with the ability to catalyze the hydrolysis of L-ascorbic acid-2-phosphate, produced ascorbic acid, which inhibited Crystals 2020, 10, 357 7 of 12 the oxidation of TMB. A fluorescence recovery signal was observed. This dual-readout sensor for ALP activity detection was 0.26 and 0.16 mU/mL by colorimetric and fluorescent methods, respectively. Qu et al. reported a ratiometric fluorescent assay for ALP detection, based on the combination of the GSH-AuNCs and SiNPs [47]. The aggregation-induced emission of AuNCs was quenched by KMnO 4 and the addition of ALP generated ascorbic acid could reduce KMnO 4 to Mn 2+ . The ratiometric fluorescence of AuNCs and SiNPs enabled the detection of ALP.

AuNCs for In Vivo Tumor Imaging
AuNCs were good candidates for the agents of metabolism studies in cells and tumor imaging in vivo, due to their distinctive properties: good stability, large Stokes shift, easy modification, good biocompatibility, low intrinsic toxicity, etc. [24]. AuNCs had the capability to combine diverse biomolecules on one system and simultaneously achieve high resolution and sensitivity, which could give more detailed anatomical or biological information [17].
Lu et al. presented a fluorescent dual-emission sensor for living cell imaging, as displayed in Figure 4 [54]. GSH-templated AuNCs (GSH-AuNCs) with oligoarginine linker peptide were decorated with silica particles via streptavidin-biotin interaction. The fluorescence intensity of GSH-AuNCs could be sensitively and selectively quenched by highly reactive oxygen species (hROS), enabling the bioimaging in live cells. Gao et al. demonstrated a facile one-pot strategy for synthesizing fluorescent AuNCs, by using 2-mercapto-5-benzimidazolesulfonic acid (MBISA) as both a protecting and reducing reagent [55]. The MBISA-AuNCs had been applied in hydrogen sulfide detection, cell imaging and pH sensing, which showed multifunctional application in life sciences. limit of 2.0 nM. This platform also allowed for the determination of glutathione reductase (GR) activity, through controlling the production of GSH [45]. Ni et al. showed an AuNCs-based dualchannel assay for turn-on fluorescent and colorimetric sensing of alkaline phosphatase (ALP) [46]. Colorless 3,3′,5,5′-tetramethylbenzidine (TMB) was oxidized into blue product (oxTMB) by BSA-AuNCs with peroxidase-like activity. The production of oxTMB induced the quenching effect of the AuNCs. The addition of ALP, with the ability to catalyze the hydrolysis of L-ascorbic acid-2phosphate, produced ascorbic acid, which inhibited the oxidation of TMB. A fluorescence recovery signal was observed. This dual-readout sensor for ALP activity detection was 0.26 and 0.16 mU/mL by colorimetric and fluorescent methods, respectively. Qu et al. reported a ratiometric fluorescent assay for ALP detection, based on the combination of the GSH-AuNCs and SiNPs [47]. The aggregation-induced emission of AuNCs was quenched by KMnO4 and the addition of ALP generated ascorbic acid could reduce KMnO4 to Mn 2+ . The ratiometric fluorescence of AuNCs and SiNPs enabled the detection of ALP.

AuNCs for in Vivo Tumor Imaging
AuNCs were good candidates for the agents of metabolism studies in cells and tumor imaging in vivo, due to their distinctive properties: good stability, large Stokes shift, easy modification, good biocompatibility, low intrinsic toxicity, etc. [24]. AuNCs had the capability to combine diverse biomolecules on one system and simultaneously achieve high resolution and sensitivity, which could give more detailed anatomical or biological information [17].
Lu et al. presented a fluorescent dual-emission sensor for living cell imaging, as displayed in Figure 4 [54]. GSH-templated AuNCs (GSH-AuNCs) with oligoarginine linker peptide were decorated with silica particles via streptavidin-biotin interaction. The fluorescence intensity of GSH-AuNCs could be sensitively and selectively quenched by highly reactive oxygen species (hROS), enabling the bioimaging in live cells. Gao et al. demonstrated a facile one-pot strategy for synthesizing fluorescent AuNCs, by using 2-mercapto-5-benzimidazolesulfonic acid (MBISA) as both a protecting and reducing reagent [55]. The MBISA-AuNCs had been applied in hydrogen sulfide detection, cell imaging and pH sensing, which showed multifunctional application in life sciences.  Attaching multiple types of ligands (small molecules, peptides, oligonucleotides, antibodies, etc.) to the AuNCs surface facilitates the achievement of tumor targeting. For instance, folic acid is commonly used for recognizing folate receptors, which are over-expressed in diverse tumor cells. Ding et al. demonstrated an AuNCs-based fluorescence biosensor for targeted imaging in cancer cells [56]. As shown in Figure 5, the combination of fluorescein and folic acid on BSA-AuNCs enabled the pH determination and targeted imaging of folate acceptor-rich cancer cells simultaneously.
Hyaluronic acid (HA), a well-known marker for cancer cells, can bind with the receptor CD44 in the membrane of the tumor cell. Zhang et al. reported BSA-AuNCs for tumor-targeted imaging in vivo [57]. The uptake of AuNCs by both cancer cells and tumor-bearing mice can be improved by the modification of FA and HA. Chen et al. presented a near-infrared fluorescent dye conjugated BSA-AuNCs for tumor imaging. The decoration of FA increased the tumor targeting capability of AuNCs [58]. Similarly, Pyo et al. synthesized FA-functionalized GSH-stabilized AuNCs for bioimaging [59].
Another common ligand is cyclic RGD (cRGD), which has the ability to bind αvβ3integrins, overexpressed on the surface of tumor cells. Additionally, aptamer AS1411 has a high affinity against nucleolin, over-expressed in the nucleus and cytoplasm of tumor cells. Dual targeting fluorescent AuNCs for bio-imaging and tissue therapy were presented by Chen et al [60]. As shown in Figure 6, cRGD and aptamer AS1411 were used to recognize proteins in the surface of tumor cell and in the nucleus inside of tumor cells, respectively, which greatly enhanced the specificity to tumors. In addition, AuNCs-cRGD-Aptamer was decorated with near infrared fluorescence dye and doxorubicin, giving a fluorescent dual-targeting probe for tumor imaging and tissue therapy. This nanoplatform displayed low cytotoxicity and tumor-targeting capability at both the in vitro and in vivo levels. This assay showed potential for clinical application in tumor imaging. Another common ligand is cyclic RGD (cRGD), which has the ability to bind α v β 3 integrins, over-expressed on the surface of tumor cells. Additionally, aptamer AS1411 has a high affinity against nucleolin, over-expressed in the nucleus and cytoplasm of tumor cells. Dual targeting fluorescent AuNCs for bio-imaging and tissue therapy were presented by Chen et al [60]. As shown in Figure 6, cRGD and aptamer AS1411 were used to recognize proteins in the surface of tumor cell and in the nucleus inside of tumor cells, respectively, which greatly enhanced the specificity to tumors. In addition, AuNCs-cRGD-Aptamer was decorated with near infrared fluorescence dye and doxorubicin, giving a fluorescent dual-targeting probe for tumor imaging and tissue therapy. This nanoplatform displayed low cytotoxicity and tumor-targeting capability at both the in vitro and in vivo levels. This assay showed potential for clinical application in tumor imaging. Hyaluronic acid (HA), a well-known marker for cancer cells, can bind with the receptor CD44 in the membrane of the tumor cell. Zhang et al. reported BSA-AuNCs for tumor-targeted imaging in vivo [57]. The uptake of AuNCs by both cancer cells and tumor-bearing mice can be improved by the modification of FA and HA. Chen et al. presented a near-infrared fluorescent dye conjugated BSA-AuNCs for tumor imaging. The decoration of FA increased the tumor targeting capability of AuNCs [58]. Similarly, Pyo et al. synthesized FA-functionalized GSH-stabilized AuNCs for bioimaging [59].
Another common ligand is cyclic RGD (cRGD), which has the ability to bind αvβ3integrins, overexpressed on the surface of tumor cells. Additionally, aptamer AS1411 has a high affinity against nucleolin, over-expressed in the nucleus and cytoplasm of tumor cells. Dual targeting fluorescent AuNCs for bio-imaging and tissue therapy were presented by Chen et al [60]. As shown in Figure 6, cRGD and aptamer AS1411 were used to recognize proteins in the surface of tumor cell and in the nucleus inside of tumor cells, respectively, which greatly enhanced the specificity to tumors. In addition, AuNCs-cRGD-Aptamer was decorated with near infrared fluorescence dye and doxorubicin, giving a fluorescent dual-targeting probe for tumor imaging and tissue therapy. This nanoplatform displayed low cytotoxicity and tumor-targeting capability at both the in vitro and in vivo levels. This assay showed potential for clinical application in tumor imaging.